No abstract

Sequence extension of the scaffoldin gene cluster from Ruminococcus flavefaciens revealed a new gene (scaE) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in Escherichia coli and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulosebound Ruminococcus flavefaciens protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in R. flavefaciens differs from those reported thus far for any other cellulosome system.

Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein.Proteomic analysis showed that the native CttA protein (ϳ130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.Ruminococcus flavefaciens is an important anaerobic, cellulolytic bacterium found in the rumen and hindgut of domestic and wild mammals (13, 17, 19, 22-24, 34, 35). Our understanding of the enzyme systems responsible for degradation of plant cell wall polymers by R. flavefaciens has advanced recently with the identification of cellulosome-like high-molecular-weight enzyme complexes (12,25,26,27). In R. flavefaciens 17, the structural protein ScaA is known to interact via cohesindockerin pairings with a range of catalytic polypeptides possessing xylanase, endoglucanase, and esterase activities (2,18,26,28). An additional structural protein, ScaC, acts as an adaptor scaffoldin, binding via its own dockerin to ScaA but also, via an unusual cohesin, to a range of other, so far unidentified proteins (27). ScaA, in turn, connects to a large structural protein, ScaB, which was recently shown to mediate attachment of the cellulosome to the bacterial cell surface through its interaction with the cell wall-anchored protein ScaE (25).It has long been recognized that noncatalytic carbohydratebinding modules (CBMs) play an important role in the initial stages of degradation of crystalline cellulose (8,29). CBMs enhance the hydrolysis of cellulose by targeting and increasing the effective concentration of either individual glycoside hydrolases or multienzyme cellulosome complexes on the surfaces of carbohydrate substrate...

In the present study, the comet, or single-cell, gel electrophoresis assay was adapted for use with the ubiquitous unicellular protozoan Tetrahymena thermophila, and the method was evaluated for its ability to detect DNA damage induced by known genotoxins and wastewater samples. The original comet assay protocol was substantially modified (e.g., lower concentrations of detergents were used in the lysis buffer; electrophoresis time was reduced). Using the modified method, T. thermophila were subjected to short exposures of phenol, hydrogen peroxide, and formaldehyde, leading to concentration-dependent increases in DNA damage. The genotoxic potential of influent and effluent water samples from a local municipal wastewater treatment plant was evaluated. The results indicated that the influent wastewater was genotoxic and that the genotoxicity in the effluent water was substantially reduced. We assume employing T. thermophila in the use of the comet assay may become a cost-effective and reliable tool for genotoxicity screening and monitoring of wastewater and similar systems.

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO(2)) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.